The world is suffering under a huge burden of obesity and type 2 diabetes. The discovery of new pathways controlling systemic energy homeostasis is badly needed. Adipose tissue is the major depot for lipid storage but also can become inflamed during obesity, contributing to certain dysfunctions in obesity. Brown fat dissipates chemical energy in the form of heat and represents an endogenous pathway to improve metabolic diseases. The past term of this MERIT award have allowed the discovery of key molecules and pathways in several aspects of adipose cell biology. We showed previously that PPAR? gets modified at serine 273 and this phosphorylation is both a cause of metabolic dysfunction in vivo and a key target for the PPAR? ligand drugs. We show here that we have created a germ-line mutation in mice whereby serine 273 of PPAR? is replaced by alanine, rendering it non-phosphorylatable. This mutant strain of animals represents a crucial opportunity to test the role of this modification in normal basal physiology and its role in obesity and insulin resistance. It also allows us to ask how much of the therapeutic effects of the PPAR? ligand drugs occur via the reduction in this phosphorylation event. New data shows that PPAR? docks the coregulator Thrap 3 only when it is phosphorylated at S273. We will study this interaction, using a variety of biochemical and biophysical techniques, including hydrogen-deuterium exchange. Importantly, we will create a series of deletions and mutations in both molecules that will better define this functional interface. This will also allow the creation of dominant negative alleles of Thrap 3. Th Thrap 3 /PPAR? interaction will be disrupted transgenically, either through the use of tissue-selective expression of a dominant-negative version of Thrap 3 or by making a tissueselective knock-out at the Thrap 3 locus. Animals bearing such transgenes will be perturbed with diet induced obesity, and analyzed by both MRI and hyperinsulinemic-euglycemic clamps. Our previous work has highlighted the crucial role that the coregulator PRDM16 plays in the fate and function of brown and beige adipose cells. In preliminary data, we have purified PRDM16 complexes from cultures enriched in beige fat cells and identified potentially interesting molecules, including LSD1, a lysine specific demethylase with a wellstudied pharmacology. We will identify important molecules in the PRDM16 complexes, especially those that distinguish beige and brown cells. To do this, we will purify PRDM16 complexes and perform comparative, quantitative proteomics by Mass Spectrometry after isobaric tagging. The function of proteins identified will be altered genetically by both gain and loss of function methods, in vitro and in vivo. Enzymes present in these complxes will be probed chemically, where the pharmacology is developed, as in the case of LSD1. The overall goal of this project will be to elucidate pathways of basic science that can inform novel therapeutic approaches to metabolic diseases such as obesity, diabetes and hepatic steatosis.